Luminescence-based immunoassays such as FOCI and LOCI have proved to be exceptionally sensitive and versatile homogeneous immunoassay techniques capable of detection of materials (analytes) such as nucleic acids, antigens, antibodies, other receptors and small molecules. Although detection in the range of several hundreds of thousand of molecules per milliliter is routinely accessible, it has not been possible to further increase the sensitivity. Nevertheless there remains a need for assays with enhanced sensitivity that can detect as few as 100 molecules per milliliter. Such improved assays would be particularly important in the detection of microbial antigens or nucleic acids which may indicate an infection when as little as one molecule is present. Other applications of the improved assay would include the detection of cytokines and other intercellular messengers that are increasingly implicated as playing a role in disease processes.
In developing an assay there are many considerations. One consideration is the signal response to changes in the concentration of an analyte. A second consideration is the ease with which the protocol for the assay may be carried out. A third consideration is the variation in interference from sample to sample. Ease of preparation and purification of the reagents, availability of equipment, ease of automation and interaction with material of interest are some of the additional considerations in developing a useful assay.
One broad category of techniques involves the use of a receptor which can specifically bind to a particular spacial and polar organization of a labeled ligand as a function of the presence of an analyte. The observed effect of binding by the receptor will depend upon the label. In some instances the binding of the receptor merely provides for a differentiation in molecular weight between bound and unbound labeled ligand. In other instances the binding of the receptor will facilitate separation of bound labeled ligand from free labeled ligand or it may affect the nature of the signal obtained from the label so that the signal varies with the amount of receptor bound to labeled ligand. A further variation is that the receptor is labeled and the ligand unlabeled. Alternatively, both the receptor and ligand are labeled or different receptors are labeled with two different labels, whereupon the labels interact when in close proximity and the amount of ligand present affects the degree to which the labels of the receptor may interact.
In specific binding assays, one molecule, usually the analyte, often serves as a bridge to cause the association of two receptors such as antibodies or DNA probes to form a complex. The complex is detected by having a label attached to at least one of the receptors. The unbound labeled receptor can be physically removed and the remaining label can be measured or the complex can be detected without separation by using a homogeneous immunoassay method. In homogeneous immunoassays involving small molecules, it is unnecessary to separate the bound and unbound label have previously been described for small molecules and thus the assay is conducted in a single or homogenous reaction mixture. These assays include SYVA's FRAT® assay, EMIT® assay, enzyme channeling immunoassay, and fluorescence energy transfer immunoassay (FETI); enzyme inhibitor immunoassays (Hoffmann LaRoche and Abbott Laboratories): fluorescence polarization immunoassay (Dandlicker), among others. All of these methods have limited sensitivity, and only a few including FETI and enzyme channeling, are suitable for large multiepitopic analytes. In contrast to homogeneous immunoassays, heterogeneous immunoassays require a separation step and are generally useful for both small and large molecules. Various labels have been used including enzymes (ELISA), fluorescent labels (FIA), radiolabels (RIA), chemiluminescent labels (CLA), etc.
In both the homogeneous and heterogeneous methods, the signal is limited by the number of labels that can be attached to the receptor and considerable effort has been expended to create structures that have many labels. In LOCI, multiple chemiluminescer molecules are incorporated into latex particles. In enzyme channeling immunoassays, multiple enzymes are attached to a particle. In heterogeneous immunoassays multiple labels are often assembled by creating avidin-biotin complexes. Many biotin molecules are attached to the antibody and avidin that is bound to multiple labels is allowed to bind to the biotin molecules. In the Chiron b-DNA assay, a complex tree-like structure is created by using branched DNA as the receptor. Each of the branches of the DNA then serves as a binder for complementary DNA that is attached to an enzyme. In all of these methods, the signal that is produced is limited by the choice of the particular label. The greatest success has been with enzyme and chemiluminescent labels. See U.S. Pat. Nos. 4,230,797; 4,238,565; 4,279,992; 4,318,981; 4,318,982; 5,710,264; Li et al. “Homogeneous Substrate-labeled Fluorescent Immunoassay for Theophylline ins Serum,” Clin. Chem., Vol. 27, pp. 22-26 (1981); and Carrico et al., “Specific Protein-Binding Reactions Monitored with Ligand-ATP Conjugates and Firefly Luciferase,” Anal. Biochem., Vol. 76, pp. 95-110 (1976).
Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth. The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means. If the label is a fluorescent molecule, the medium can be irradiated and the fluorescence determined. Where the label is a radioactive group, the medium can be counted to determine the radioactive count.
When very low concentrations of analyte must be detected as required for small clinical samples, the current methods are slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable. A method for increasing the sensitivity to permit the use of simple, rapid, nonisotopic, homogeneous or heterogeneous methods for detecting analytes is therefore desirable.